Method and pharmaceutical compositions for drug targeting

ABSTRACT

A pharmaceutical composition comprising an oil/water emulsion wherein the oil droplets contain a drug in dissolved or dispersed or solubilized form. The droplets are further coated with adsorbed native or modified antibodies which provide targeting of the droplets and the drug. The process for preparing this composition comprises the steps of (i) dissolving or dispersing a drug in an oil phase, (ii) preparing an oil/water emulsion, (iii) obtaining surface-active antibodies by chemical or physical attachment of hydrophobic groups to the antibodies, and (iv) mixing the surface-active antibody with the emulsion.

The present invention relates to pharmaceutical compositions and amethod for drug targeting. More specifically said invention relates topharmaceutical compositions containing an oil/water emulsion wherein adrug is dissolved or solubilized or dispersed inside the oil dropletsand wherein said emulsion droplets are coated with adsorbed native andmodified antibodies. The invention relates also to a process for thepreparation of said pharmaceutical compositions and to a method for drugtargeting toward specific molecules or sites in the body, comprisingadministration to a host an effective amount of above mentionedpharmaceutical compositions.

Emulsion droplets are coated with adsorbed native and/or modifiedantibodies which are capable of interacting with specific molecules orantigenic determinants. Therefore, the droplets will be directed towardsspecific molecules or sites in the body. When a drug is dissolved,dispersed or solubilized inside the oil droplet, a novel drug targetingsystem is obtained.

BACKGROUND OF THE INVENTION

The present invention relates to drug targeting by small emulsiondroplets, using biologically active targeting molecules. The advantagesof being able to direct the drug to the tissue or cells where it isrequired, and to minimize the amount delivered to inappropriate siteshas implications for many clinical situations, such as cancerchemotherapy, inflammations and viral infections (Davis S. S et al. DrugExptl. Clin. Res. 9, 632, 1985).

In the past, several attempts to achieve drug targeting were reported,by using polyclonal and monoclonal antibodies. These attempts include:

1. Direct chemical attachment of drug molecules to an antibody molecule.

2. Chemical attachment of drugs to antibodies through the use of alinkage polymer molecule such as dextran.

3. Attachment of small antibodies to small biodegradable polymericparticles, by covalent linkage by direct adsorption or by adsorption viaprotein A.

4. Coupling of liposomes with monoclonal antibodies via hydrophobicmodification of the antibody.

The above suggested method to achieve drug targeting have manydisadvantages:

a) Covalent attachment of drug molecules require development of achemical binding process for each drug to be tested.

b) Direct attachment to the antibody may reduce its biological activity.

c) only a limited amount of drug molecules may be bound.

d) The clinical effects of the drugs may be altered upon chemicalattachment.

e) Possible leakage of drugs if liposomes are used.

f) Desorption of the antibodies may occur if it is physically adsorbedto a solid particle.

The present invention will provide a novel drug targeting method, whichwould overcome most of the above disadvantages, by the use of emulsionsand micro emulsions.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions containingan oil/water emulsion wherein a drug is dissolved or dispersed orsolubilized inside the oil droplets and wherein said emulsion dropletsare coated with adsorbed native and/or modified antibodies.

The present invention further relates to a process for the preparationof said pharmaceutical compositions comprising:

a) dissolving the desired drug in an oil phase;

b) formation of an oil/water emulsion;

c) formation of a surface-active-antibody by chemical attachment ofvarious hydrophobic groups to the antibodies;

d) mixing the surface-active antibody with the emulsion.

The present invention also relates to a method for drug targetingtowards specific molecules or sites in the body (such as antigenicdeterminants), comprising administration to a host of an effectiveamount of pharmaceutical composition according to claim 1.

DESCRIPTION OF THE INVENTION

The invention is based on a simple process, which may be applied tovarious types of drugs.

As described in FIG. 1, the final composition contains oil emulsiondroplets onto which native or chemically modified antibodies areadsorbed. A hydrophobic drug is dissolved or solubilized inside the oildroplet, and there-fore, the drug may be targeted to specific sites bythe antibodies. The following principle steps are required for obtainingthe final composition:

1. Chemical attachment of various hydrophobic groups to the antibodies.This step will lead to formation of a "surface-active-antibody".

2. Formation of an O/W emulsion by various simple methods which are wellestablished. The oil phase initially contains the desired drugs.

3. Mixing the "surface-active-antibody" with the emulsion, for a shortperiod of time. The resulting composition is demonstrated in FIG. 1.

This process is very simple and may be applied rapidly to various typesof drugs, antibodies, and emulsions. The main advantages of the proposedmethod are:

1. The process is very versatile and is based on a modular approach,which may be adopted by the final user, provided the drug has a suitablesolubility in the emulsion droplets, or may be dispersed or solubilizedin the oil phase.

2. It is possible to use various types of oils for preparation of theemulsion to meet the requirement of drug solubility or dispersion.

3. The drug molecules are not subjected to any chemical modification;the original drug is maintained through the whole process.

4. Due to its hydrophobicity, the drug will not leak significantly fromthe oil droplets upon storage.

5. Desorption of the antibodies is a very unlikely event since theantibody has become a "surface-active-antibody" with improved adsorptioncapability; more protein molecules are adsorbed more strongly to theoil-water interface, than the native antibody.

6. The chemical modification of the antibodies is very simple and isperformed in such a way that the biological activity and antigenrecognition is not affected.

7. The same process may be applied to other biologically activesubstances which have a recognition capability.

8. The same process may be applied even without modification of theantibody by the use of specific molecules such as protein A.

9. The same process may be applied by physical attachment of hydrophobicgroups to the antibody without covalent bonding.

10. The emulsion droplets serve as large reservoirs for drugs, and bythe antibodies it is possible to obtain high local drug concentrationwithout side effects.

Manner and Process of Making the Invented Composition

The apparatus and materials disclosed herein are merely exemplary, andafter understanding the method, other embodiments may be devised.

Step I: Emulsion Preparation

1. The desired drug is dissolved or dispersed in the oil phase, whichmight be soybean oil, medium chain triglycerides (MCT) or any other oil,with increased or decreased polarity and hydrophobicity. The oil maycontain additions such as solubilizers, dispersants, etc.

2. An emulsifier (such as lecithin and pluronic F-68, or a combinationof emulsifiers) is dissolved in an aqueous phase.

3. The oil phase is dropwise added to the aqueous phase while stirred bya mechanical or magnetic stirrer.

4. The crude emulsion is further homogenized until the desired dropletsize is achieved. This step may be carried out by various instruments,such as polytron (Brinkman Instruments), ultra-torex (Jumble & Kundel),high pressure piston homogenizers, microfluidizer, etc. The wholeprocess for preparation of the emulsion may take less than half an hour.Typical composition contains about 20% W/W oil phase, 1-5% emulsifiersand water or saline up to 100%.

Step II: Antibody Modification

The desired antibody (monoclonal or polyclonal) is coupled to ahydrophobic tail by a simple chemical reaction. During this step someparameters may vary such as the ratio of hydrophobic tails to antibodymolecules and the length of the hydrophobic tail. It is important tonote that only a slight modification is needed to impose surfaceactivity without decreasing the biological activity.

The process described here is based on the use of active esters of fattyacids, but other methods may also be applied, and also physicaladsorption of various groups.

The principle steps are:

1. Formation of N-alkanoyl succinimide ester (active ester) by reactinga fatty acid (chain lengths C8-C18) with N-hydroxy succinimide, asdescribed by Lapidot et al. J. Lipid Research, 8, 142 (1967)!.

2. Mixing the active ester with a solution of the desired antibodies,and formation of alkanoyl antibody, as described by Huang et al. J.Biological Chem., 235, 8015-8018 (1980)!.

3. Purification of the modified antibody by dialysis and/or Sephadexcolumn.

The whole process is simple and requires no special equipment. Thereaction conditions are chosen in such a way that the biologicalactivity will not be affected, as will be described in the examples.

Step III: Antibody-Emulsion Interaction

Since the modified antibodies are very surface active, all is needed fortheir adsorption onto emulsion droplet is simple mixing for about onehour.

The modified antibody solution may be mixed with various volumes of theemulsion. (In some cases it is also possible to mix native and modifiedantibodies before the adsorption take place.)

After the adsorption process is completed, the final composition isachieved. This composition contains strongly attached antibodies at theoil-water interface and is capable of recognizing specific sites such asvirally infected cells or cancer cells, depending on the type ofantibody used.

EXAMPLES

1. Modification of IgG with N-caproyl ester was conducted at variousester/IgG ratios. The resulting antibodies were surface-active, havingvarious numbers of attached hydrophobic groups.

After modification was completed, the biological activity was analyzedby quantitative evaluation of the active antibodies against HerpesSimplex Virus by two methods:

Elisa, and fluorescence antibody assay labeling virally infected cells.From the results presented in FIG. 2 it is clear that up to a molarratio of 4:1, the recognition activity is not affected. The same resultsare obtained from the recognition of virally infected BSC-1 cells, aspresented in FIG. 3. Similar results were obtained for infected Daudicells (from human origin).

2. A series of modified and native antibodies were adsorbed ontohydrophobic silica particles in order to evaluate the increase in thesurface activity. As shown in FIG. 4, the amount of adsorbed antibodyincreases with the increase of the ratio ether/antibody.

3. The hydrophobic particles, which have adsorbed antibodies, weresubjected to several washings with Phosphate Buffer Saline (PBS) inorder to evaluate their desorption. As shown in FIG. 5, the nativeantibody is readily desorbed while almost no desorption was detected forthe modified antibodies.

4. A series of native and modified IgG were adsorbed on O/W wateremulsions, and their effective activity was evaluated. Emulsioncompositions was: 10% W/W soybean oil, 2% W/O lipoid PC and 88% W/Wphosphate buffer saline. The adsorption process is based on mixing 1 mlemulsion with 7.3 mg antibody and PBS up to a final volume of 19 ml. Theingredients were gently mixed for 12 hrs at 37° C. The detection ofactivity was achieved by addition of FITC labeled anti IgG (H+C), mixingand reading the fluorescence after adsorption. It is clear that if themodified IgG will lose its activity, no fluorescence will be detected onthe emulsion droplets. The results obtained by a computerizedfluorescence microscope indicated, as shown in FIG. 6, that enhancementof activity is indeed obtained with IgG which was modified at a lowester/antibody ratio.

5. The attachment of the antibody may be achieved by adsorption ofprotein A to the emulsion droplet. This could lead to selectiveadsorption of the antibody with its Fab fragments facing the externalaqueous phase. The new system: emulsion-protein A(native ormodified)-antibody is a unique modular and versatile system in which nopreparations are needed for each drug-antibody combination.

6. The purpose of the following example is to demonstrate the targetingof emulsion droplets to specific cells by in-vitro experiments.

An oil-in-water emulsion containing an oil soluble fluorescent markerwas prepared. Onto this emulsion a modified antibody was adsorbed. (Theantibody was modified by attachment of C₈. residues of fatty acid, 10%of the total available amine groups.)

The emulsion which contained the adsorbed antibody was added to twoplates (30 minutes) which were covered by either normal BSC-1 cells, orBSC-1 cells which were previously infected by Herpes virus (HSV-1).After rinsing the plates it was found by a fluorescence microscope andalso by ordinary microscope that emulsion droplets (coated withantibodies) were found only on the infected cells, as presented in FIG.7.

As a control, a similar experiment was conducted, but without adsorbedantibodies. After rinsing the plates, emulsion droplets were notdetected at all.

This example shows that the emulsion droplets having adsorbed antibodiesagainst Herpes virus could be attached specifically onto Herpes infectedcells.

We claim:
 1. A pharmaceutical composition comprising an oil-in-wateremulsion wherein the oil emulsion droplet contains dissolved ordispersed or solubilized a hydrophobic drug and on its surface absorbedantibodies which are modified by attaching hydrphobic groups whereinsaid composition is made by a process comprising the following steps:a)solubilizing, dissolving or dispersing the desired drug in an oil; b)forming an oil-in-water emulsion the oil being present in an amount toform oil droplets that contain the desired drug; c) forming separately asurface-active-antibody by chemical or physical attachment of varioushydrophobic groups to the antibodies; and d) mixing the surface-activeantibody with the oil-in-water emulsion that contains the desired drugin the oil.
 2. The pharmaceutical composition according to claim 1wherein the oil is of soybean oil, triglycerides, or any other oil withhydrophobic molecules.
 3. The pharmaceutical composition according toclaim 1 wherein the drug is hydrophobic.
 4. The pharmaceuticalcomposition according to claim 1 wherein the drug is useful for cancerchemotherapy, inflammations, and infectious diseases including fungi andviral infections.
 5. A process for preparing a pharmaceuticalcomposition which comprises an oil-in-water emulsion wherein the oilemulsion droplet contains dissolved or dispersed or solubilized ahydrophobic drug and on its surface adsorbed antibodies which aremodified by attaching hydrophobic groups which comprises:a)solubilizing, dissolving or dispersing the desired drug in an oil; b)forming an oil-in-water emulsion the oil being present in a amount toform oil droplets that contain the desired drug; c) forming separately asurface-active-antibody by chemical or physical attachment of varioushydrophobic groups to the antibodies; and d) mixing the surface-activeantibody with the oil-in-water emulsion that contains the desired drugin the oil.